IGF-1R antibody and its use for the diagnosis of cancer

ABSTRACT

The present disclosure relates to a novel antibody, in particular a monoclonal antibody, capable of binding to IGF-1R, as well as the amino and nucleic acid sequences coding for said antibody.

The present invention relates to a novel antibody, in particular amonoclonal antibody, capable of binding to IGF-1R, as well as the aminoand nucleic acid sequences coding for said antibody.

The insulin-like growth factor 1 receptor called IGF-1R (or sometimesIGF1R) is a receptor with tyrosine kinase activity having 70% homologywith the insulin receptor IR. IGF-1R is a glycoprotein of molecularweight approximately 350,000. It is a hetero-tetrameric receptor ofwhich each half—linked by disulfide bridges—is composed of anextracellular α-subunit and of a transmembrane β-subunit. IGF-1R bindsIGF1 and IGF2 with a very high affinity (Kd #1 nM) but is equallycapable of binding to insulin with an affinity 100 to 1000 times lower.Conversely, the IR binds insulin with a very high affinity although theIGFs only bind to the insulin receptor with a 100 times lower affinity.The tyrosine kinase domains of IGF-1R and of IR have a very highsequence homology although the zones of weaker homology respectivelyconcern the cysteine-rich region situated on the α-subunit and theC-terminal part of the β-subunit. The sequence differences observed inthe α-subunit are situated in the binding zone of the ligands and aretherefore at the origin of the relative affinities of IGF-1R and of IRfor the IGFs and insulin respectively. The differences in the C-terminalpart of the β-subunit result in a divergence in the signalling pathwaysof the two receptors; IGF-1R mediating mitogenic, differentiation andantiapoptosis effects, while the activation of the IR principallyinvolves effects at the level of the metabolic pathways.

The role of the IGF system in carcinogenesis has become the subject ofintensive research in the last 20 years. This interest followed thediscovery of the fact that in addition to its mitogenic andantiapoptosis properties, IGF-1R seems to be required for theestablishment and the maintenance of a transformed phenotype. In fact,it has been well established that an overexpression or a constitutiveactivation of IGF-1R leads, in a great variety of cells, to a growth ofthe cells independent of the support in media devoid of foetal calfserum, and to the formation of tumors in nude mice. This in itself isnot a unique property since a great variety of products of overexpressedgenes can transform cells, including a good number of receptors ofgrowth factors. However, the crucial discovery which has clearlydemonstrated the major role played by IGF-1R in the transformation hasbeen the demonstration that the IGF-1W cells, in which the gene codingfor IGF-1R has been inactivated, are totally refractory totransformation by different agents which are usually capable oftransforming the cells, such as the E5 protein of bovine papillomavirus, an overexpression of EGFR or of PDGFR, the T antigen of SV 40,activated Ras or the combination of these two last factors.

In such a context IGF-1R has been considered for a long time as aninteresting target in oncology. A large number of projects targetingIGF-1R (humanized or human antibodies or small molecules) have beeninitiated to develop IGF-1R antagonists for the treatment of cancers andmore than 70 clinical trials have been performed in various indications.Nevertheless, at this date, none of these projects have been successfuland there are no IGF-1R antibodies on the market.

The present invention aims to provide at least one reagent that can beused as a diagnostic or prognosis biomarker for detecting and/ormonitoring oncogenic disorders especially those characterized byexpression of IGF-1R or those that are mediated by aberrant IGF-1Rexpression.

Previous attempts to develop a valuable antibody that can be used as arelevant diagnostic or prognostic tool have been reported but none ofthese are giving satisfaction.

As it will be apparent from the following examples, the inventors havebeen surprised to demonstrate that the commercial antibodies commonlyused at this day for the scoring of the IGF-1R expressing tumors seem tobe not relevant as they give false positive and/or false negative. Thisissue has lead, in part, to the failure of clinical trials with IGF-1Rantibodies due to the selection of the patients rather than the realactivity of the IGF-1R antibodies.

Moreover, first studies performed using commercial antibodies showeddiscrepancy between IGF-1R scoring and anti-tumoral activity of targetedADC therapy.

The present invention intends to remedy this issue by providing a novelantibody which, contrary to the existing ones, is capable of strainingwhich do correlate with the pharmacology of IGF-1R targeted therapy.

In a first aspect, a subject of the invention is an isolated antibody,or an antigen-binding fragment thereof, that binds to the IGF-1R,preferably human IGF-1R, with high affinity and can thus be useful inmethods to diagnose pathological hyperproliferative oncogenic disordersmediated by IGF-1R expression.

An embodiment of the invention relates to an antibody, or anantigen-binding fragment thereof, comprising the six CDRs of sequencesSEQ ID Nos. 1, 2, 3, 4, 5 and 6.

In a particular embodiment, the invention relates to an IGF-1R antibody,or an antigen-binding fragment thereof, characterized in that itcomprises:

i) a heavy chain with CDR-H1 of sequence SEQ ID No. 1, CDR-H2 ofsequence SEQ ID No. 2 and CDR-H3 of sequence SEQ ID No. 3; and

ii) a light chain with CDR-L1 of sequence SEQ ID No. 4, CDR-L2 ofsequence SEQ ID No. 5 and CDR-L3 of sequence SEQ ID No. 6.

The terms “antibody”, “antibodies” “ab” or “immunoglobulin” are usedinterchangeably in the broadest sense and include monoclonal antibodies,isolated, engineered, chemically synthesized, or recombinant antibodies(e.g., full length or intact monoclonal antibodies), polyclonalantibodies, multivalent antibodies or multispecific antibodies (e.g.,bispecific antibodies) and also antibody fragment, so long as theyexhibit the desired biological activity. In an embodiment, the inventionrelates to a recombinant antibody.

As used in the present specification, the expression “IGF-1R antibody”should be interpreted as similar to “anti-IGF-1R antibody” and means anantibody capable of binding to IGF-1R.

By “IGF-1R binding fragment” or “antigen-binding fragment” of anantibody, it is intended to indicate any peptide, polypeptide, orprotein retaining the ability to bind to the IGF-1R target (alsogenerally referred as antigen) of the antibody. In an embodiment, such“antigen binding fragments” are selected in the group consisting of Fv,scFv (sc for single chain), Fab, F(ab′)₂, Fab′, scFv-Fc fragments ordiabodies, or any fragment of which the half-life time would have beenincreased by chemical modification, such as the addition ofpoly(alkylene) glycol such as poly(ethylene) glycol (“PEGylation”)(pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab′)₂-PEG orFab′-PEG) (“PEG” for Poly(Ethylene) Glycol), or by incorporation into aliposome, said fragments having at least one of the characteristic CDRsof the antibody according to the invention. Preferably, said “antigenbinding fragments” will be constituted or will comprise a partialsequence of the heavy or light variable chain of the antibody from whichthey are derived, said partial sequence being sufficient to retain thesame specificity of binding as the antibody from which it is descendedand a sufficient affinity, preferably at least equal to 1/100, in a morepreferred manner to at least 1/10, of the affinity of the antibody fromwhich it is descended, with respect to the target.

Preferably, said “IGF-1R binding fragment” or “antigen-binding fragment”comprises at least:

i) the CDR-H1 of sequence SEQ ID No. 1, CDR-H2 of sequence SEQ ID No. 2and CDR-H3 of sequence SEQ ID No. 3; and

ii) the CDR-L1 of sequence SEQ ID No. 4, CDR-L2 of sequence SEQ ID No. 5and CDR-L3 of sequence SEQ ID No. 6.

By “binding”, “binds”, or the like, it is intended that the antibody, orany antigen-binding fragment thereof, forms a complex with an antigenthat is relatively stable under physiologic conditions. Specific bindingcan be characterized by an equilibrium dissociation constant of at leastabout 1×10⁻⁶ M or less. Methods for determining whether two moleculesbind are well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like. For the avoidance ofdoubt, it does not mean that the said antibody could not bind orinterfere, at a low level, to another antigen. Nevertheless, as anembodiment, the said antibody binds only to the said antigen.

By CDR regions or CDR(s), it is intended to indicate the hypervariableregions of the heavy and light chains of the immunoglobulins as definedby IMGT.

The IMGT unique numbering has been defined to compare the variabledomains whatever the antigen receptor, the chain type, or the species[Lefranc M.-P., Immunology Today 18, 509 (1997)/Lefranc M.-P., TheImmunologist, 7, 132-136 (1999)/Lefranc, M.-P., Pommié, C., Ruiz, M.,Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. andLefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT uniquenumbering, the conserved amino acids always have the same position, forinstance cystein 23 (1st-CYS), tryptophan 41 (CONSERVED-TRP),hydrophobic amino acid 89, cystein 104 (2nd-CYS), phenylalanine ortryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides astandardized delimitation of the framework regions (FR1-IMGT: positions1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to128) and of the complementarity determining regions: CDR1-IMGT: 27 to38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps representunoccupied positions, the CDR-IMGT lengths (shown between brackets andseparated by dots, e.g. [8.8.13]) become crucial information. The IMGTunique numbering is used in 2D graphical representations, designated asIMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics,53, 857-883 (2002)/Kaas, Q. and Lefranc, M.-P., Current Bioinformatics,2, 21-30 (2007)], and in 3D structures in IMGT/3Dstructure-DB [Kaas, Q.,Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data.Nucl. Acids. Res., 32, D208-D210 (2004)].

It must be understood that, without contradictory specification in thepresent specification, complementarity-determining regions or CDRs, meanthe hypervariable regions of the heavy and light chains ofimmunoglobulins as defined according to the IMGT numbering system.

Nevertheless, CDRs can also be defined according to the Kabat numberingsystem (Kabat et al., Sequences of proteins of immunological interest,5^(th) Ed., U.S. Department of Health and Human Services, NIH, 1991, andlater editions). There are three heavy-chain CDRs and three light-chainCDRs. Here, the terms “CDR” and “CDRs” are used to indicate, dependingon the case, one or more, or even all, of the regions containing themajority of the amino acid residues responsible for the antibody'sbinding affinity for the antigen or epitope it recognizes. In order tosimplify the reading of the present application, the CDRs according toKabat are not defined. Nevertheless, it would be obvious for the personskilled in that art, using the definition of the CDRs according to IMGT,to define the CDRs according to Kabat.

In a particular embodiment, the IGF-1R antibody according to theinvention is characterized in that it comprises a heavy chain variabledomain of sequence SEQ ID No. 7, or any sequence with at least 90% ofhomology with the sequence SEQ ID No. 7.

In a particular embodiment, the IGF-1R antibody according to theinvention is characterized in that it comprises a light chain variabledomain of sequence SEQ ID No. 8, or any sequence with at least 90% ofhomology with the sequence SEQ ID No. 8.

According to still another embodiment, the antibody referred as 810D12,is characterized in that it comprises a heavy-chain variable domainsequence comprising the amino acid sequence SEQ ID No. 7 or a sequencewith at least 80%, preferably 85%, 90%, 95% and 98% of homology afteroptimal alignment with sequence SEQ ID No. 7; and/or in that itcomprises a light-chain variable domain sequence comprising the aminoacid sequence SEQ ID No. 8 or a sequence with at least 80%, preferably85%, 90%, 95% and 98% of homology after optimal alignment with sequenceSEQ ID No. 8.

In the sense of the present invention, the “percentage of homology”between two sequences of nucleic acids or amino acids means thepercentage of identical nucleotides or amino acid residues between thetwo sequences to be compared, obtained after optimal alignment, thispercentage being purely statistical and the differences between the twosequences being distributed randomly along their length. The comparisonof two nucleic acid or amino acid sequences is traditionally carried outby comparing the sequences after having optimally aligned them, saidcomparison being able to be conducted by segment or by using an“alignment window”. Optimal alignment of the sequences for comparisoncan be carried out, in addition to comparison by hand, by means of thelocal homology algorithm of Smith and Waterman (1981) [Ad. App. Math.2:482], by means of the local homology algorithm of Neddleman and Wunsch(1970) [J. Mol. Biol. 48:443], by means of the similarity search methodof Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444] or bymeans of computer software using these algorithms (GAP, BESTFIT, FASTAand TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis., or by the comparison softwareBLAST NR or BLAST P). For the amino acid sequence exhibiting at least80%, preferably at least 85%, 90%, 95% and 98% of homology with areference amino acid sequence, preferred examples include thosecontaining the reference sequence, certain modifications, notably adeletion, addition or substitution of at least one amino acid,truncation or extension. In the case of substitution of one or moreconsecutive or non-consecutive amino acids, substitutions are preferredin which the substituted amino acids are replaced by “equivalent” aminoacids. Here, the expression “equivalent amino acids” is meant toindicate any amino acids likely to be substituted for one of thestructural amino acids without however modifying the biologicalactivities of the corresponding antibodies and of those specificexamples defined below.

Equivalent amino acids can be determined either on their structuralhomology with the amino acids for which they are substituted or on theresults of comparative tests of biological activity between the variousantigen binding proteins likely to be generated.

As a non-limiting example, table 1 below summarizes the possiblesubstitutions likely to be carried out without resulting in asignificant modification of the biological activity of the correspondingmodified antigen binding protein; inverse substitutions are naturallypossible under the same conditions.

TABLE 1 Original residue Substitution(s) Ala (A) Val, Gly, Pro Arg (R)Lys, His Asn (N) Gln Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly(G) Ala His (H) Arg Ile (I) Leu Leu (L) Ile, Val, Met Lys (K) Arg Met(M) Leu Phe (F) Tyr Pro (P) Ala Ser (S) Thr, Cys Thr (T) Ser Trp (W) TyrTyr (Y) Phe, Trp Val (V) Leu, Ala

A particular aspect of the invention is that the antibody, or anyantigen binding fragment thereof, does not bind to the Insulin receptor(IR).

In another embodiment, the antibody of the invention consists of amonoclonal antibody.

The term “monoclonal antibody” or “Mab” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e. the individual antibodies of the population areidentical except for possible naturally occurring mutations that may bepresent in minor amounts. Monoclonal antibodies are highly specific,being directed against a single epitope. Such monoclonal antibody may beproduced by a single clone of B cells or hybridoma. Monoclonalantibodies may also be recombinant, i.e. produced by proteinengineering. Monoclonal antibodies may also be isolated from phageantibody libraries. In addition, in contrast with preparations ofpolyclonal antibodies which typically include various antibodiesdirected against various determinants, or epitopes, each monoclonalantibody is directed against a single epitope of the antigen. Theinvention relates to an antibody isolated or obtained by purificationfrom natural sources or obtained by genetic recombination or chemicalsynthesis.

In another embodiment, the antibody of the invention consists of arecombinant antibody. The term “recombinant antibody” refers to anantibody that results from the expression of recombinant DNA withinliving cells. A recombinant antibody of the invention is obtained byusing laboratory methods of genetic recombination, well known by aperson skilled in the art, creating DNA sequences that would not befound in biological organisms.

In another embodiment, the antibody of the invention consists of achemically synthesized antibody.

“IGF-1R antibody” includes (without contrary specification) the murine,but also the chimeric and the humanized forms of the said IGF-1Rantibody.

For more clarity, the following table 2 illustrates the sequences of theantibody 810D12, defined according to IMGT.

TABLE 2 CDR SEQ Antibody numbering Heavy chain Light chain ID NO.810D12I- IMGT CDR-H1 1 4893 CDR-H2 2 CDR-H3 3 CDR-L1 4 CDR-L2 5 CDR-L3 6variable domain 7 variable domain 8

In one embodiment, the monoclonal antibody herein includes murine,chimeric and humanized antibody. The antibody can be derived from anhybridoma of murine origin filed within the French collection formicroorganism cultures (CNCM, Pasteur Institute, Paris, France), saidhybridoma being obtained by the fusion of Balb/C immunized micesplenocytes/lymphocytes and cells of the myeloma Sp 2/O—Ag 14 cell line.

According to another aspect, the invention relates to a murine hybridomacapable of secreting a monoclonal antibody according to the invention,notably the hybridoma of murine origin deposited at the CNCM, InstitutPasteur, Paris, France, on Sep. 17, 2014, under the number 1-4893.

The monoclonal antibody, here referred as 810D12, or any antigen-bindingfragment thereof, being secreted by the said hybridoma 1-4893 obviouslyforms part of the present invention.

The invention relates to an IGF-1R antibody, or an antigen-bindingfragment thereof, characterized in that it is secreted by the hybridomafiled at the CNCM, Institut Pasteur, Paris, on Sep. 17, 2014, undernumber 1-4893.

The invention also describes the murine hybridoma filed at the CNCM,Institut Pasteur, Paris, on Sep. 17, 2014, under number 1-4893.

A novel aspect of the present invention relates to an isolated nucleicacid, characterized in that it is chosen from the following nucleicacids:

-   -   a) a nucleic acid coding for an antibody according to the        invention;    -   b) a nucleic acid comprising a sequence selected from the        sequences SEQ ID No.9 or 10, or a sequence with at least 80%,        preferably 85%, 90%, 95% and 98% of homology after optimal        alignment with the sequences SEQ ID No. 9 or 10; and    -   e) a complementary nucleic acids of the nucleic acids as defined        in a) or b).

Table 3 below summarizes the various nucleotide sequences concerning theantibody 810D12 of the invention.

TABLE 3 Antibody Heavy chain Light chain SEQ ID NO. 810D12I- variabledomain 9 4893 variable domain 10

The terms “nucleic acid”, “nucleic sequence”, “nucleic acid sequence”,“polynucleotide”, “oligonucleotide”, “polynucleotide sequence” and“nucleotide sequence”, used interchangeably in the present description,mean a precise sequence of nucleotides, modified or not, defining afragment or a region of a nucleic acid, containing unnatural nucleotidesor not, and being either a double-strand DNA, a single-strand DNA ortranscription products of said DNAs.

It should also be included here that the present invention does notrelate to nucleotide sequences in their natural chromosomal environment,i.e., in a natural state. The sequences of the present invention havebeen isolated and/or purified, i.e., they were sampled directly orindirectly, for example by a copy, their environment having been atleast partially modified. Isolated nucleic acids obtained by recombinantgenetics, by means, for example, of host cells, or obtained by chemicalsynthesis should also be mentioned here.

The invention also relates to a vector comprising a nucleic acid asdescribed in the invention.

The invention notably targets cloning and/or expression vectors thatcontain such a nucleotide sequence.

The vectors of the invention preferably contain elements which allow theexpression and/or the secretion of nucleotide sequences in a given hostcell. The vector thus must contain a promoter, translation initiationand termination signals, as well as suitable transcription regulationregions. It must be able to be maintained in a stable manner in the hostcell and may optionally have specific signals which specify secretion ofthe translated protein. These various elements are selected andoptimized by a person skilled in the art according to the host cellused. For this purpose, the nucleotide sequences can be inserted inself-replicating vectors within the chosen host or be integrativevectors of the chosen host.

Such vectors are prepared by methods typically used by a person skilledin the art and the resulting clones can be introduced into a suitablehost by standard methods such as lipofection, electroporation, heatshock or chemical methods.

The vectors are, for example, vectors of plasmid or viral origin. Theyare used to transform host cells in order to clone or express thenucleotide sequences of the invention.

The invention also comprises host cells transformed by or comprising avector as described in the present invention.

The host cell can be selected among prokaryotic or eukaryotic systemssuch as bacterial cells, for example, but also yeast cells or animalcells, notably mammal cells. Insect or plant cells can also be used.

The invention also relates to animals, other than man, that have atransformed cell according to the invention.

Another aspect of the invention relates to a method for the productionof an antibody according to the invention, or one of its functionalfragments, characterized in that said method comprises the followingsteps:

a) the culture in a medium of and the suitable culture conditions for ahost cell according to the invention; and

b) the recovery of said antibody, or one of its functional fragments,thus produced from the culture medium or from said cultured cells.

The transformed cells according to the invention are of use in methodsfor the preparation of recombinant polypeptides according to theinvention. Methods for the preparation of polypeptide according to theinvention in recombinant form, characterized in that said methods use avector and/or a cell transformed by a vector according to the invention,are also comprised in the present invention. Preferably, a celltransformed by a vector according to the invention is cultured underconditions that allow the expression of the aforesaid polypeptide andrecovery of said recombinant peptide.

As already mentioned, the host cell can be selected among prokaryotic oreukaryotic systems. In particular, it is possible to identify thenucleotide sequences of the invention that facilitate secretion in sucha prokaryotic or eukaryotic system. A vector according to the inventioncarrying such a sequence can thus be used advantageously for theproduction of recombinant proteins to be secreted. Indeed, thepurification of these recombinant proteins of interest will befacilitated by the fact that they are present in the supernatant of thecellular culture rather than inside host cells.

The use of the antibody of the invention as biomarker is also disclosed.The methods may be used for detecting or diagnosing varioushyperproliferative oncogenic disorders associated with expression ofIGF-1R exemplified by, but not limited to, prostate cancer,osteosarcomas, lung cancer, breast cancer, endometrial cancer,glioblastoma, colon, cancer, gastric cancer, renal cancer, pancreascancer, head and neck cancer or any other cancer associated withexpression of IGF-1R. As would be recognized by one of ordinary skill inthis art, the level of antibody expression associated with a particulardisorder will vary depending on the nature and/or the severity of thepre-existing condition.

Administration of the antibodies of the present invention in any of theconventional ways known to one skilled in the art (e.g., topical,parenteral, intramuscular, etc.), will provide an extremely usefulmethod of detecting dysplastic cells in a sample as well as allowing aclinician to monitor the therapeutic regiment of a patient undergoingtreatment for a hyperproliferative disorder associated with or mediatedby expression of IGF-1R.

The antibody of the invention, or an antigen-binding fragment thereof,will find use in various medical or research purposes, including thedetection, diagnosis, prognosis and staging of various pathologiesassociated with expression of IGF-1R.

An embodiment of the invention relates to the IGF-1R antibody, or anantigen-binding fragment thereof, as above described for use as an agentfor the detection of IGF-1R expressing tumoral cells.

Another embodiment of the invention is the IGF-1R antibody, or anantigen-binding fragment thereof, as above described, for use in the invitro or ex vivo diagnosing or prognosing of an oncogenic disorderassociated with expression of IGF-1R.

“Diagnosing” a disease as used herein refers to the process ofidentifying or detecting the presence of a pathologicalhyperproliferative oncogenic disorder associated with or mediated byexpression of IGF-1R, monitoring the progression of the disease, andidentifying or detecting cells or samples that are indicative of adisorder associated with the expression of IGF-1R.

“Prognosis” as used herein means the likelihood of recovery from adisease or the prediction of the probable development or outcome of adisease. For example, if a sample from a subject is negative forstaining with the IGF-1R antibody, then the “prognosis” for that subjectis better than if the sample is positive for IGF-1R staining. Samplesmay be scored for IGF-1R expression levels on an appropriate scale as itwill be more detailed hereinafter.

The IGF-1R antibody can be present in the form of an immunoconjugate orof a labeled-antibody to obtain a detectable/quantifiable signal. Whenused with suitable labels or other appropriate detectable biomoleculesor chemicals, the IGF-1R antibody is particularly useful for in vitroand in vivo diagnosis and prognosis applications.

Labels for use in immunoassays are generally known to those skilled inthe art and include enzymes, radioisotopes, and fluorescent, luminescentand chromogenic substances, including colored particles such ascolloidal gold or latex beads. Suitable immunoassays includeenzyme-linked immunosorbent assays (ELISA). Various types of labels andmethods of conjugating the labels to the IGF-1R antibodies are wellknown to those skilled in the art, such as the ones set forth below.

As used herein, the term “an oncogenic disorder associated withexpression of IGF-1R” is intended to include diseases and otherdisorders in which the presence of high levels of IGF-1R (aberrant) in asubject suffering from the disorder has been shown to be or is suspectedof being either responsible for the pathophysiology of the disorder or afactor that contributes to a worsening of the disorder. Alternatively,such disorders may be evidenced, for example, by an increase in thelevels of IGF-1R on the cell surface in the affected cells or tissues ofa subject suffering from the disorder. The increase in IGF-1R levels maybe detected using the IGF-1R antibody.

In certain embodiments, “increased expression” as it relates to IGF-1Rrefers to protein or gene expression levels that demonstrate astatistically significant increase in expression (as measured by RNAexpression or protein expression) relative to a control.

An embodiment is an IGF-1R antibody, or an antigen-binding fragmentthereof, as above described, for use in determining whether a patientwith an oncogenic disorder is likely to benefit from treatment with aninhibitor targeting the IGF-1R pathway, preferentially an IGF-1Rantibody alone, combined or conjugated.

As used in the present specification, the expression “inhibitortargeting the IGF-1R pathway” means any compound capable of decreasingor inhibiting the tyrosine kinase activity of IGF-1R, either by bindingto the ligand(s) of IGF-1R or to the IGFR itself. Examples for suchinhibitors are protein, peptides, antibodies or Antibody-Drug-Conjugatesor any chemical compound which act as IGF-1R antagonists, antisenseoligonucleotides or siRNA inhibiting expression of the IGF-1R gene or ofa gene encoding one of the IGFR ligand(s), or any other drug or compoundknown by the person skilled in the art.

More particularly, in the sense of the present specification, theinhibitor targeting the IGF-1R pathway is intended to encompass anycompound or molecule capable of binding to the IGF-1R and inhibiting thebinding of its ligand(s).

Still more particularly, in the sense of the present specification, theinhibitor targeting the IGF-1R pathway is intended to encompass anymonoclonal antibody which binds to the IGF-1R.

In another preferred embodiment, the inhibitor targeting the IGF-1Rpathway consists of an Antibody-Drug-Conjugate (ADC) wherein theantibody moiety targets the IGF-1R and the Drug moiety can be selectedfrom any drugs such as cytotoxic, cytostatic, toxins, etc. . . . . In anexemplified embodiment, the drug moiety can consist of an auristatin, ananalog or a derivative.

It is also an object of the invention to describe a method for detectingin vitro or ex vivo the presence and/or the location of IGF-1Rexpressing tumoral cells in a subject, said method comprising the stepsof:

(a) contacting a biological sample from the said subject with the IGF-1Rantibody, or an antigen-binding fragment thereof, according to thepresent invention as above described; and

(b) detecting the binding of the said IGF-1R antibody, or anantigen-binding fragment thereof, with the said biological sample.

The present invention is also directed to an in vitro or ex vivo methodfor detecting and/or to quantify and/or to determine the level of, theexpression of IGF-1R in, preferably at the surface of cells of, asubject, said method comprising the steps of:

(a) contacting a biological sample from the said subject with the IGF-1Rantibody, or an antigen-binding fragment thereof, according to thepresent invention as above described; and

(b) detecting, and/or quantifying, and/or determining the level of, thebinding of the said IGF-1R antibody, or an antigen-binding fragmentthereof, with the said biological sample.

The binding of the IGF-1R antibody may be detected and/or quantifiedand/or determined by various assays available to the skilled artisan.Although any suitable means for carrying out the assays are includedwithin the invention, Fluorescence Activated Cell Sorting (FACS), ELISA,western blotting and immunohistochemistry (IHC) can be mentioned inparticular. Preferred methods include IHC and FACS.

The invention also describes a method for detecting in vitro or ex vivothe percentage of tumoral cells expressing IGF-1R in a subject, saidmethod comprising the steps of:

(a) contacting a biological sample from the said subject with the IGF-1Rantibody, or an antigen-binding fragment thereof, as above described;and

(b) quantifying the percentage of cells expressing IGF-1R in thebiological sample.

Another embodiment is a method for determining in vitro or ex vivo theexpression level of IGF-1R in tumoral cells or in a tumor in a subject,said method comprising the steps of:

(a) contacting a biological sample from the said subject with the IGF-1Rantibody, or an antigen-binding fragment thereof, as above described;and

(b) quantifying the level of binding of the said IGF-1R antibody, or anantigen-binding fragment thereof, to IGF-1R in the said biologicalsample.

As will be apparent to the skilled artisan, the level of IGF-1R antibodybinding to IGF-1R may be quantified by any means known to the person ofskills in the art. Preferred methods involve the use of immunoenzymaticprocesses, such as ELISA assays, immunofluorescence, IHC,radio-immunoassay (RIA), or FACS.

According to the method of the invention, the level of binding of thesaid IGF-1R antibody, or an antigen-binding fragment thereof, to IGF-1Ris quantified by Fluorescence Activated Cell Sorting (FACS) orimmunohistochemistry (IHC).

A “biological sample” may be any sample that may be taken from asubject. Such a sample must allow for the determination of theexpression levels of the biomarker of the invention. The nature of thesample will thus be dependent upon the nature of the tumor.

Preferred biological samples include samples such as a blood sample, aplasma sample, or a lymph sample, if the cancer is a liquid tumor.

Preferred biological samples include samples such as a biopsy sample ora sample taken from a surgical resection therapy, if the cancer is asolid tumor.

Preferably, the biological sample is a biological fluid, such as serum,whole blood cells, a tissue sample or a biopsy of human origin. Thesample may for example include, biopsied tissue, which can beconveniently assayed for the presence of a pathological oncogenicdisorder associated with expression of IGF-1R.

Once a determination is made of the IGF-1R expression level in thetested biological samples, the results can be compared with those ofcontrol samples, which are obtained in a manner similar to the testedbiological samples but from individuals that do not have an oncogenicdisorder associated with expression of IGF-1R. If the level of IGF-1R issignificantly elevated in the tested biological sample, it may beconcluded that there is an increased likelihood of the subject fromwhich it was derived has or will develop said disorder.

The invention relates to a process of in vitro or ex vivo diagnosis orprognosis of an IGF-1R expressing tumor, wherein said process comprisesthe steps of (i) determining the expression level of IGF-1R by the amethod for determining in vitro or ex vivo the expression level ofIGF-1R in tumoral cells or in a tumor in a subject according to thepresent invention and as above described, and (ii) comparing theexpression level of step (i) with a reference expression level of IGF-1Rfrom normal tissue or a non expressing IGF-1R tissue.

With regards to the development of targeted antitumor therapy, thediagnosis with immunohistological techniques gives in situ informationon the receptor expression level and thus enables to select patientssusceptible to be treated following the expression level of receptorsneeded for such treatment.

Stage determination has potential prognosis value and provides criteriafor designing optimal therapy. Simpson et al., J. Clin. Oncology 18:2059(2000). For example, treatment selection for solid tumors is based ontumor staging, which is usually performed using theTumor/Node/Metastasis (TNM) test from the American Joint Committee onCancer (AJCC). It is commonly acknowledged that, while this test andstaging system provides some valuable information concerning the stageat which solid cancer has been diagnosed in the patient, it is impreciseand insufficient. In particular, it fails to identify the earlieststages of tumor progression.

Another embodiment consists of a method for determining in vitro or exvivo the IGF-1R scoring of tumoral cells or of the tumor in a subject,said method comprising the steps of:

(a) contacting a biological sample from the said subject with the IGF-1Rantibody, or an antigen-binding fragment thereof, as above described;

(b) quantifying by Fluorescence Activated Cell Sorting (FACS) orimmunohistochemistry (IHC) the level of binding of the said IGF-1Rantibody, or an antigen-binding fragment thereof, to IGF-1R in the saidbiological sample; and

(c) scoring the tumoral cells or the tumor by comparing the quantifiedlevel obtained in step (b) to an appropriate scale based on twoparameters which are the intensity of the staining and the percentage ofpositive cells.

In an embodiment, the IGF-1R antibody is capable of binding IGF-1R whentissue samples are, formalin fixed-, formol substituted fixed-,Glyco-fixx fixed-, paraffin embedded and/or frozen.

Any conventional hazard analysis method may be used to estimate theprognostic value of IGF-1R. Representative analysis methods include Coxregression analysis, which is a semiparametric method for modelingsurvival or time-to-event data in the presence of censored cases (Hosmerand Lemeshow, 1999; Cox, 1972). In contrast to other survival analyses,e.g. Life Tables or Kaplan-Meyer, Cox allows the inclusion of predictorvariables (covariates) in the models. Using a convention analysismethod, e.g., Cox one may be able to test hypotheses regarding thecorrelation of IGF-1R expression status of in a primary tumor totime-to-onset of either disease relapse (disease-free survival time, ortime to metastatic disease), or time to death from the disease (overallsurvival time). Cox regression analysis is also known as Coxproportional hazard analysis. This method is standard for testing theprognostic value of a tumor marker on patient survival time. When usedin multivariate mode, the effect of several covariates are tested inparallel so that individual covariates that have independent prognosticvalue can be identified, i.e. the most useful markers. The term negativeor positive “IGF-1R status” can also be referred as [IGF-1R (−)] or[IGF-1R (+)].

A sample may be “scored” during the diagnosis or monitoring of cancer.In its simplest form, scoring may be categorical negative or positive asjudged by visual examination of samples by immunohistochemistry. Morequantitative scoring involves judging the two parameters intensity ofstaining and the proportion of stained (“positive”) cells that aresampled.

“IGF-1R status” within the meaning of the invention, relates to theclassification of tumor to a IGF-1R positive [IGF-1R (+)] or IGF-1Rnegative [IGF-1R (−)] class based on the determination of the expressionlevel of the IGF-1R as measured by any methods such asimmunohistochemistry (IHC), Fluorescence Activated Cell Sorting FACS, orother methods known by the person skilled in the art.

In an embodiment, to ensure standardization, samples may be scored forIGF-1R expression levels on different scales, most of them being basedon an assessment of the intensity of the reaction product and thepercentage of positive cells (Payne et al., Predictive markers in breastcancer—the present, Histopathology 2008, 52, 82-90).

In another embodiment, said scoring, particularly in step (c) of themethod of the present invention, comprises using an appropriate scalebased on the intensity of the staining and the percentage of positivecells.

As a first example, by analogy with the Quick Allred scoring for IHCassessment of oestrogen receptor and progesterone receptor, samples maybe scored for IGF-1R expression levels on a global scale from 0 to 8combining scores for intensity of reactivity and for the proportion ofcells stained (Harvey J M, Clarck G M, Osborne C K, Allred D C; J. Clin.Oncol. 1999; 17; 1474-1481). More particularly, the first criteria ofintensity of reactivity is scored on a scale from 0 to 3, 0corresponding to “No reactivity” and 3 corresponding to “Strongreactivity”. The second criteria of proportion reactive is scored on ascale from 0 to 5, 0 corresponding to “No reactivity” and 5 to “67-100%proportion reactive”. The intensity of the reactivity score and theproportion reactive score are then summed to produce total score of 0through 8. A total score of 0-2 is regarded as negative while a totalscore of 3-8 is regarded as positive.

According to this scale, the terms negative or positive “IGF-1R status”of tumors or of tumoral cells used in the present description refers tolevels of expression of IGF-1R that correspond to scores 0-2 or 3-8 onthe Allred scale, respectively.

Table 4 hereinafter illustrates the guidelines for interpreting IHCresults according to Allred method.

TABLE 4 Intensity of immunoreactivity Score 1 Proportion reactive Score2 No reactivity 0 No reactivity 0 Weak reactivity 1   <1% 1 Moderatereactivity 2  1-10% 2 Strong reactivity 3 11-33% 3 — 34-66% 4 — 67-100% 5 Total Score (Score 1 + Score 2) Interpretation 0-2 Negative 3-8Positive

According to the invention, the method is characterized in that the saidappropriate scale is a scale of 0 to 8 wherein no reactivity is scored0, and a strong reactivity in a proportion of 67-100% proportionreactive is scored 8.

Thus, in a preferred embodiment, the method for determining in vitro orex vivo the IGF-1R scoring of tumoral cells or of a tumor in a subjectaccording to the present invention, is characterized in that in step (c)the said appropriate scale is a scale of 0 to 8 wherein no reactivity isscored 0, and a strong reactivity in a proportion of 67-100% proportionreactive is scored 8.

In other words, it is described and claimed a process of determining invitro or ex vivo the status of a tumor or of tumoral cells from asubject, wherein said process comprises the steps of:

-   (a) scoring a tumor or of tumoral cells from a subject according to    the Allred scale; and-   (b) i) determining that the status of the tumor or of tumoral cells    is [IGF-1R(+)] with an Allred score of 3 to 8; or

ii) determining that the status of the tumor or of tumoral cells is[IGF-1R(−)] with an Allred score of 0 to 2.

In a particular aspect of the invention, the status of the tumor or oftumoral cells is [IGF-1R (+)] with an Allred score of 3.

In a particular aspect of the invention, the status of the tumor or oftumoral cells is [IGF-1R (+)] with an Allred score of 4.

In a particular aspect of the invention, the status of the tumor or oftumoral cells is [IGF-1R (+)] with an Allred score of 5.

In a particular aspect of the invention, the status of the tumor or oftumoral cells is [IGF-1R (+)] with an Allred score of 6.

In a particular aspect of the invention, the status of the tumor or oftumoral cells is [IGF-1R (+)] with an Allred score of 7.

In a particular aspect of the invention, the status of the tumor or oftumoral cells is [IGF-1R (+)] with an Allred score of 8.

In another particular aspect of the invention, the status of the tumoror of tumoral cells is [IGF-1R (+)] with an Allred score of 3 to 8.

Another particular method herein described for determining in vitro orex vivo the IGF-1R status of tumoral cells or of the tumor in a subject,is characterized in that it comprises the steps of:

(a) scoring IGF-1R tumoral cells or of the tumor from the said subjectaccording to the method of the claim 18; and

(b) determining that the IGF-1R status of tumoral cells or of the tumoris [IGF-1R(+)] with a score of 3 to 8; or

(c) determining that the IGF-1R status of tumoral cells or of the tumoris [IGF-1R(−)] with a score of 0 to 2.

As a second example, by analogy with the conventional scoring for IHCassessment of HER-2 receptor for example, samples may be scored forIGF-1R expression levels on a somewhat simpler scoring methodintegrating the intensity of staining (preferentially membranousstaining) and the proportion of cells that display staining into acombined scale from 0 to 3+.

In this scale, referred as the simplified scale, 0 and 1+ are negativewhereas 2+ and 3+ represents positive staining. Nevertheless, scores1+-3+ can be recoded as positive because each positive score may beassociated with significantly higher risk for relapse and fatal diseasewhen compared to score 0 (negative), but increasing intensity among thepositive scores may provide additional risk reduction.

Generally speaking, the terms negative or positive “IGF-1R status” oftumors or of tumoral cells used in the present description refers tolevels of expression of IGF-1R that correspond to scores 0-1+ or 2+-3+on the simplified scale, respectively. Only complete circumferentialmembranous reactivity of the invasive tumor should be considered andoften resembled a “chicken wire” appearance. Under current guidelines,samples scored as borderline (score of 2+ or 3+) for IGF-1R are requiredto undergo further assessment. The IHC analysis should be rejected, andeither repeated or tested by FISH or any other method if, as nonlimitative example, controls are not as expected, artifacts involve mostof the sample and the sample has strong membranous positivity of normalbreast ducts (internal controls) suggesting excessive antigen retrieval.

For more clarity, table 5 hereinafter summarizes these parameters.

TABLE 5 IGF-1R status IHC description 0 No reactivity or membranousreactivity in less than 10% of tumour cells 1⁺ Faint/barely perceptiblemembranous reactivity is detected in more than 10% of tumour cells. Thecells are immunoreactive only in part of the membrane. 2⁺ Weak tomoderate complete membranous reactivity is seen in more than 10% oftumour cells. 3⁺ Strong complete reactivity is seen in more than 10% oftumour cells.

The method of the invention is characterized in that the saidappropriate scale is a scale of 0 to 3⁺ wherein no membranous reactivityof tumor cells is scored 0 and strong complete reactivity in more than10% of tumor cells is scored 3⁺.

In more details, as above described, said appropriate scale is a scaleof 0 to 3 wherein no membranous reactivity of tumor cells is scored 0;faint perceptible membranous reactivity in more than 10% of tumor cellsis scored 1+; weak to moderate complete membranous reactivity in morethan 10% of tumor cells is scored 2+; and strong complete reactivity inmore than 10% of tumor cells is scored 3+.

In other words, it is described and claimed a process of determining invitro or ex vivo the status of a tumor of of tumoral cells from asubject, wherein said process comprises the steps of (a) scoring a tumoror tumoral cells from a subject according to the simplified scale asabove described; and (b) determining that the status of the tumor or oftumoral cells is [IGF-1R(+)] with a score of 2+ or 3+; or (c)determining that the status of the tumor or of tumoral cells is[IGF-1R(−)] with a score of 0 or 1+.

In a particular aspect of the invention, a tumor or tumoral cells is[IGF-1R (+)] with a score of 2+.

In a particular aspect of the invention, a tumor is, or tumoral cellsare [IGF-1R (+)] with a score of 3+.

In another particular aspect of the invention, a tumor is, or tumoralcells are [IGF-1R (+)] with a score of 2+ or 3+.

In another embodiment, the invention relates to a method for determiningin vitro or ex vivo the IGF-1R status tumoral cells or a tumor in asubject, said method comprising the steps of:

-   (a) scoring said IGF-1R tumoral cells or said tumor from the said    subject according to the method of the present invention described    before; and-   (b) i) determining that the IGF-1R status of tumoral cells or of the    tumor is [IGF-1R(+)] with a score of 2⁺ or 3⁺; or

ii) determining that the IGF-1R status of tumoral cells is [IGF-1R(−)]with a score of 0 or 1⁺.

Generally, the results of a test or assay can be presented in any of avariety of formats. The results can be presented qualitatively. Forexample, the test report may indicate only whether or not a particularpolypeptide was detected, perhaps also with an indication of the limitsof detection. The results may be displayed as semi-quantitative. Forexample, various ranges may be defined, and the ranges may be assigned ascore (e.g., 0 to 3+ or 0 to 8 depending on the used scale) thatprovides a certain degree of quantitative information. Such a score mayreflect various factors, e.g., the number of cells in which IGF-1R isdetected, the intensity of the signal (which may indicate the level ofexpression of IGF-1R or IGF-1R-bearing cells), etc. The results may bedisplayed in a quantitative way, e.g., as a percentage of cells in whichIGF-1R is detected, as a protein concentration, etc.

As will be appreciated by one of ordinary skill in the art, the type ofoutput provided by a test will vary depending upon the technicallimitations of the test and the biological significance associated withdetection of the polypeptide. For example, in the case of certainpolypeptides a purely qualitative output (e.g., whether or not thepolypeptide is detected at a certain detection level) providessignificant information. In other cases a more quantitative output(e.g., a ratio of the level of expression of the polypeptide in thesample being tested versus the normal level) is necessary.

In another aspect, it is described a method of diagnosing pathologicalhyperproliferative oncogenic disorder or a susceptibility to apathological condition associated with expression of IGF-1R in asubject, said method comprising the steps of:

(a) determining the presence or absence of IGF-1R carrying cells in asample by a method for the detection of IGF-1R expressing cells and/orfor determining the level of expression of IGF-1R according to thepresent invention, and

(b) diagnosing a pathological condition or susceptibility to apathological condition based on the presence or absence of said IGF-1Rbearing cells.

In the methods herein described, the detection of IGF-1R expressingcells or an increase in the levels of IGF-1R is generally indicative ofa patient with or suspected of presenting a IGF-1R mediated disorder.

The present invention also provides a method for predicting the risk ofan individual to develop a cancer, said method comprising detecting theexpression level of IGF-1R in a tissue sample by a method for thedetection of IGF-1R expressing cells and/or for determining the level ofexpression of IGF-1R according to the present invention, wherein a highlevel of IGF-1R expression is indicative of a high risk of developing acancer.

The invention also relates to a method for evaluating tumoraggressiveness.

“Tumor aggressiveness” as used herein refers to a tumor quickly growingand tending to spread rapidly.

In one embodiment, the said method for evaluating tumor aggressivenesscomprises the step of:

(a) determining the level of IGF-1R expressed by cells in a tumorsample, by a method for the detection of IGF-1R expressing cells and/orfor determining the level of expression of IGF-1R according to thepresent invention,

(b) determining the level of IGF-1R expressed in an equivalent tissuesample taken from the same individual at a later time by a method forthe detection of IGF-1R expressing cells and/or for determining thelevel of expression of IGF-1R according to the present invention, and

(c) determining the ratio between the expression level obtained in step(a) and the ratio obtained in step (b)

wherein the ratio of IGF-1R expression in the tumor sample over timeprovides information on the risks of cancer progression.

In a preferred embodiment, a ratio of the level obtained in step (a) tothe level obtained in step (b) greater than 1 indicates aggressiveness.In another embodiment, a ratio inferior or equal to 1 indicates nonaggressiveness.

Another aspect of the invention is the monitoring of IGF-1R expressionin response to the administration of a therapy targeting the IGF-1Rpathway by involving the method for the detection of, and/or to quantifyIGF-1R, and/or to determine the level of, expression according to thepresent invention. Such a monitoring can be very useful when the saidtherapy triggers the downregulation and/or the degradation of IGF-1R.

It is also an object of the invention to describe a method fordetermining whether an oncogenic disorder is susceptible to treatmentwith an antibody drug targeting the IGF-1R pathway, said methodcomprising the steps of:

(a) determining in vitro or ex vivo the IGF-1R status of tumoral cellsof a tumor of a subject according to the method of scoring of thepresent invention as above described, and

(b) determining that, if the IGF-1R status of tumoral cells or of thetumor is IGF-1R(+), the oncogenic disorder is susceptible to treatmentwith an antibody drug targeting the IGF-1R pathway.

In particular, monitoring IGF-1R expression on the cell surface could bea critical tool for evaluating the efficacy of the treatment duringclinical trials and “personalized” therapies.

The application thus provides methods for determining the appropriatetherapeutic regimen for a subject.

An increase or a decrease in the level of IGF-1R which can be determinedby the method for the detection of and/or to determine the level of,expression according to the present invention, is indicative of theevolution of a cancer associated with IGF-1R. Thus, by measuring anincrease in the number of cells expressing IGF-1R or changes in theconcentration of IGF-1R present in various tissues or cells, it ispossible to determine whether a particular therapeutic regime aimed atameliorating a malignancy associated with IGF-1R is effective.

Another object of the invention is also a method for determining invitro or ex vivo the efficacy of a therapeutic regimen designed toalleviate an oncogenic disorder associated with IGF-1R in a subjectsuffering from said disorder, said method comprising the steps of:

(a) determining a first expression level of IGF-1R by the method for thedetection of and/or to determine the level of, expression according tothe present invention, as above described in a first biological sample,said first biological sample corresponding to first time point of thesaid treatment;

(b) determining a second expression level of IGF-1R by the method forthe detection of and/or to determine the level of, expression accordingto the present invention, as above described in a second biologicalsample, said second biological sample corresponding to a second, latertime point of the said treatment;

(c) calculating the ratio of the said first expression level obtained instep (a) to the said second expression level obtained in step (b); and

(d) determining that the efficacy of said therapeutic regime is highwhen the ratio of step (c) is greater than 1; or determining that theefficacy of said therapeutic regime is low when the ratio of step (c) isinferior or equal to 1.

In a preferred embodiment, the said therapeutic regime designed toalleviate an oncogenic disorder associated with IGF-1R in a subjectsuffering from said disorder includes the administration of a therapytargeting the IGF-1R pathway to the said subject.

It is also an object of the invention to provide an in vivo method ofimaging an oncogenic disorder associated with expression of IGF-1R usingthe method for the detection of and/or to determine the level of,expression according to the present invention. Such a method is usefulfor localizing in vivo the tumoral cells, as well as monitoring theirinvasiveness. Likewise, the method is useful for monitoring theprogression and/or the response to treatment in patients previouslydiagnosed with a IGF-1R-mediated cancer.

An embodiment is a method for detecting the location of IGF-1Rexpressing tumoral cells in a subject, said method comprising the stepsof:

a) administering the IGF-1R antibody, or a antigen-binding fragmentthereof, according to the present invention to the subject; and

b) detecting binding of said IGF-1R antibody,

wherein said binding indicates the presence of the tumoral cells.

As for the detection of the presence of an expressing tumor, manytechniques known by the person skilled in the art can be used.Nevertheless, preferred means are IHC and FACS.

In another aspect, the invention provides an in vivo imaging reagent,the said reagent comprising the IGF-1R antibody, or an antigen-bindingfragment thereof, according to the present invention, the said IGF-1Rantibody being preferably labeled, more preferably radio labeled.

The present invention also contemplates the use of the said reagent inmedical imaging of a patient suffering from an IGF-1R-mediated cancer.

The method of the invention comprises the steps of:

(a) administering to the said patient an imaging-effective amount of animaging reagent of the invention and

(b) detecting the said reagent.

In a preferred embodiment, the imaging agent comprises the IGF-1Rantibody, or an antigen-binding fragment thereof, according to thepresent invention, and an active moiety.

An “active moiety” as used herein is an agent which permits in vivodetection of the said imaging reagent. The active moiety according tothe invention includes in particular radio-elements such asTechnetium-99m (99mTc), Copper-67 (Cu-67), Scandium-47 (Sc-47),Luthetium-77 (Lu-177) copper-64 (Cu-64), Yttrium-86 (Y-86) or Iodine-124(I-124).

The imaging agent is administered in an amount effective for diagnosticuse in a mammal such as a human and the localization and accumulation ofthe imaging agent is then detected. The localization and accumulation ofthe imaging agent may be detected by radionucleide imaging,radioscintigraphy, nuclear magnetic resonance imaging, computedtomography, positron emission tomography, computerized axial tomography,X-ray or magnetic resonance imaging method, fluorescence detection, andchemiluminescent detection.

With regards to the development of targeted antitumor therapy, thediagnosis with immunohistological techniques gives in situ informationon the receptor expression level, e.g. as regards the size and/or thelocation of the tumor. The diagnosis thus enables to select patientssusceptible to be treated following the expression level of receptorsneeded for such a treatment.

A particular interesting aspect of the invention is a method forselecting a cancer patient predicted to benefit or not from theadministration of a therapeutic amount of an antibody drug targeting theIGF-1R pathway, said method comprising the steps of:

(a) determining the expression level of IGF-1R according to the methodof the invention above described;

(b) comparing the expression level of the previous step (a) with areference expression level; and

(c) selecting the patient as being predicted to benefit from a treatmentwith an antibody drug targeting the IGF-1R pathway, if the ratio of theexpression level obtained in (a) to the reference expression level isgreater than 1; or

(d) selecting the patient as being not predicted to benefit from atreatment with an antibody drug targeting the IGF-1R pathway, if theratio of the expression level obtained in (a) to the referenceexpression level is inferior or equal to 1.

The expression level of IGF-1R is advantageously compared or measured inrelation to levels in a control cell or sample also referred to as a“reference level” or “reference expression level”. “Reference level”,“reference expression level”, “control level” and “control” are usedinterchangeably in the specification. A “control level” means a separatebaseline level measured in a comparable control cell, which is generallydisease or cancer free. The said control cell may be from the sameindividual, since, even in a cancerous patient, the tissue which is thesite of the tumor still comprises non tumor healthy tissue. It may alsooriginate from another individual who is normal or does not present withthe same disease from which the diseased or test sample is obtained.Within the context of the present invention, the term “reference level”refers to a “control level” of expression of IGF-1R used to evaluate atest level of expression of IGF-1R in a cancer cell containing sample ofa patient. For example, when the level of IGF-1R in the biologicalsample of a patient is higher than the reference level of IGF-1R, thecells will be considered to have a high level of expression, oroverexpression, of IGF-1R. The reference level can be determined by aplurality of methods. Expression levels may thus define IGF-1R bearingcells or alternatively the level of expression of IGF-1R independent ofthe number of cells expressing IGF-1R. Thus the reference level for eachpatient can be prescribed by a reference ratio of IGF-1R, wherein thereference ratio can be determined by any of the methods for determiningthe reference levels described herein.

For example, the control may be a predetermined value, which can take avariety of forms. It can be a single cut-off value, such as a median ormean. The “reference level” can be a single number, equally applicableto every patient individually, or the reference level can vary,according to specific subpopulations of patients. Thus, for example,older men might have a different reference level than younger men forthe same cancer, and women might have a different reference level thanmen for the same cancer. Alternatively, the “reference level” can bedetermined by measuring the level of expression of IGF-1R innon-oncogenic cancer cells from the same tissue as the tissue of theneoplastic cells to be tested. As well, the “reference level” might be acertain ratio of IGF-1R in the neoplastic cells of a patient relative tothe IGF-1R levels in non-tumor cells within the same patient. The“reference level” can also be a level of IGF-1R of in vitro culturedcells, which can be manipulated to simulate tumor cells, or can bemanipulated in any other manner which yields expression levels whichaccurately determine the reference level. On the other hand, the“reference level” can be established based upon comparative groups, suchas in groups not having elevated IGF-1R levels and groups havingelevated IGF-1R levels. Another example of comparative groups would begroups having a particular disease, condition or symptoms and groupswithout the disease. The predetermined value can be arranged, forexample, where a tested population is divided equally (or unequally)into groups, such as a low-risk group, a medium-risk group and ahigh-risk group.

The reference level can also be determined by comparison of the level ofIGF-1R in populations of patients having the same cancer. This can beaccomplished, for example, by histogram analysis, in which an entirecohort of patients are graphically presented, wherein a first axisrepresents the level of IGF-1R, and a second axis represents the numberof patients in the cohort whose tumor cells express IGF-1R at a givenlevel. Two or more separate groups of patients can be determined byidentification of subsets populations of the cohort which have the sameor similar levels of IGF-1R. Determination of the reference level canthen be made based on a level which best distinguishes these separategroups. A reference level also can represent the levels of two or moremarkers, one of which is IGF-1R. Two or more markers can be represented,for example, by a ratio of values for levels of each marker.

Likewise, an apparently healthy population will have a different‘normal’ range than will have a population which is known to have acondition associated with expression of IGF-1R. Accordingly, thepredetermined value selected may take into account the category in whichan individual falls. Appropriate ranges and categories can be selectedwith no more than routine experimentation by those of ordinary skill inthe art. By “elevated” “increased” it is meant high relative to aselected control. Typically the control will be based on apparentlyhealthy normal individuals in an appropriate age bracket.

It will also be understood that the controls according to the inventionmay be, in addition to predetermined values, samples of materials testedin parallel with the experimental materials. Examples include tissue orcells obtained at the same time from the same subject, for example,parts of a single biopsy, or parts of a single cell sample from thesubject.

In another embodiment, the invention relates to a pharmaceuticalcomposition for in vivo imaging of an oncogenic disorder associated withexpression of IGF-1R comprising the IGF-1R antibody, or anantigen-binding fragment thereof, according to the present inventionabove described, or an antigen binding fragment thereof, which islabeled and a pharmaceutically acceptable carrier.

In another aspect, it is also described a kit for the detection ofIGF-1R expressing tumoral cells in a patient, characterized in that saidkit comprises at least the IGF-1R antibody, or an antigen-bindingfragment thereof, as above described, and preferentially the antibody810D12.

Packaged materials comprising a combination of reagents in predeterminedamounts with instructions for performing the diagnostic assay, e.g.kits, are also within the scope of the invention. The kit contains theIGF-1R antibodies for detection and quantification of IGF-1R in vitro,e.g. in an ELISA. Where the IGF-1R antibody is labeled with an enzyme,the kit will include substrates and cofactors required by the enzyme(e.g., a substrate precursor which provides the detectable chromophoreor fluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. Such a kit may comprise a receptacle being compartmentalized toreceive one or more containers such as vials, tubes and the like, suchcontainers holding separate elements of the invention. For example, onecontainer may contain a first antibody bound to an insoluble or partlysoluble carrier. A second container may contain soluble,detectably-labeled second antibody, in lyophilized form or in solution.The receptacle may also contain a third container holding a detectablylabeled third antibody in lyophilized form or in solution. A kit of thisnature can be used in the sandwich assay of the invention. The label orpackage insert may provide a description of the composition as well asinstructions for the intended in vitro or diagnostic use.

The relative amounts of the various reagents may be varied widely toprovide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

In yet a further aspect, IGF-1R antibodies or antigen-binding fragmentsthereof as detailed herein according to the present invention, areprovided labeled with a detectable moiety, such that they may bepackaged and used, for example, in kits, to diagnose or identify cellshaving the aforementioned antigen. Non-limiting examples of such labelsinclude fluorophores such as fluorescein isothiocyanate; chromophores,radionuclides, biotine or enzymes. Such labeled IGF-1R antibodies may beused for the histological localization of the antigen, ELISA, cellsorting, as well as other immunological techniques for detecting orquantifying IGF-1R, and cells bearing this antigen, for example.

The present invention is also directed to a kit, wherein said kit ischaracterized in that it comprises an IGF-1R antibody or antigen-bindingfragments thereof, according to the present invention.

The present invention is also directed to a kit, wherein said kit ischaracterized in that it comprises a chimeric or humanized IGF-1Rantibody or antigen-binding fragments thereof, which can be obtainedfrom the 6 CDRs having the sequences SEQ ID Nos. 1 to 6 of the IGF-1Rantibody or antigen-binding fragments thereof, according to the presentinvention.

Kits are also provided that are useful as a positive control forpurification or immunoprecipitation of IGF-1R from cells. For isolationand purification of IGF-1R, the kit can contain the IGF-1R antibody orantigen-binding fragments thereof as detailed herein according to thepresent invention coupled to beads (e.g., sepharose beads). Kits can beprovided which contain the antibodies for detection and quantitation ofIGF-1R in vitro, e.g. in an ELISA. The kit comprises a container and alabel or package insert on or associated with the container. Additionalcontainers may be included that contain, e.g., diluents and buffers,control antibodies. The label or package insert may provide adescription of the composition as well as instructions for the intendedin vitro or diagnostic use.

More particularly, the invention concerns a kit for the in vitro or exvivo determination of the IGF-1R status of tumoral cells of a tumor in asubject by the methods herein described. In a preferred embodiment, asit will be described in the example, the invention relates to a kit forthe determination of the IGF-1R status of a tumor or of tumoral cells byIHC and/or FACS methods.

In a particular embodiment, the invention consists in a kit comprisingat least the IGF-1R antibody, or an antigen-binding fragment thereof, ofthe present invention as above described, said antibody being labeled.

In a preferred embodiment, the kit according to the invention furthercomprises a reagent useful for detecting the extent of binding betweenthe said IGF-1R antibody and IGF-1R.

In another preferred embodiment, the kit of the invention useful fordetermining in vitro or ex vivo the expression level of IGF-1R in aIGF-1R-expressing tumor, further comprises a reagent useful forquantifying the level of binding between the said labeled IGF-1Rantibody and IGF-1R.

In still another embodiment, the kit according to the invention furthercomprises: i) a reagent useful for detecting the extent of bindingbetween the said labeled IGF-1R antibody and IGF-1R; and ii) positiveand negative control samples useful for the scoring the IGF-1Rexpression level.

Said kit can further comprise a polyclonal antibody specific to murineantibodies or to human/humanized antibodies, preferably said polyclonalantibody specific to murine, humanized or human antibodies is labeled.

According to a particular embodiment of the invention, the kit forselecting in vitro a cancer patient who is predicted to benefit or notbenefit from therapeutic administration of an inhibitor targeting theIGF-IR pathway can comprise: i) a reagent useful for detecting theextent of binding between the said IGF-1R antibody and IGF-1R; ii)control level that has been correlated with sensitivity to a IGF-1Rinhibitor and/or iii) control level that has been correlated withresistance to a IGF-1R inhibitor.

The invention also relates to a kit for determining whether a patientwith an oncogenic disorder is likely to benefit from treatment with anantibody drug targeting the IGF-1R pathway, characterized in that saidkit comprises at least the IGF-1R antibody, or an antigen-bindingfragment thereof, of the present invention as above described.

In another embodiment, said kit according is characterized in that itfurther comprises

i) a reagent for detecting the extent of binding between the said IGF-1Rantibody and IGF-1R on the surface of tumoral cells; and/or

ii) a reagent for quantifying the level of binding between the saidIGF-1R antibody and IGF-1R on the surface of tumoral cells.

Other characteristics and advantages of the invention appear in thecontinuation of the description with the examples and the figures whoselegends are represented below.

FIG. 1: Graphic representation of OD values obtained with 810D12antibody in the rhIGF1R ELISA. Data fitting and EC₅₀ determination aredetermined using Prism application.

FIGS. 2A-2C: Immunohistochemistry (IHC) patterns of recognition ofparaffin embedded tumor MCF-7 with the 810D12 (FIG. 2A), with G11anti-IGF-1R antibody (Roche Ventana) (FIG. 2B) or AF-305 (R&D system)anti-IGF-1R antibody (FIG. 2C).

FIG. 3: In vivo activity of an anti-IGF-1R ADC in the MCF-7 xenograftmodel.

FIGS. 4A-4C: Immunohistochemistry (IHC) patterns of recognition ofparaffin embedded tumor SBC-5 with the 810D12, with G11 anti-IGF-1Rantibody (Roche Ventana) (FIG. 4B) or AF-305 (R&D system) anti-IGF-1Rantibody (FIG. 4C).

FIG. 5: In vivo activity of an anti-IGF-1R ADC in the SBC-5 xenograftmodel.

EXAMPLE 1 810D12 Generation and Selection

Mabs generated against rhIGF-1R were produced and selected as describedbelow.

Female Balb/C mice were immunized by subcutaneous injection with 10 μgof recombinant human IGF-1R protein (R and D Systems, 391-GR) withFreund Adjuvant. Immunisation was repeated three times at 2 weeksintervals. The fourth injection was made by intraperitoneal injection inpresence of adjuvant.

Three days later spleen cells were fused with SP2OAg14 myeloma cellswith PEG 50%. After 14 days of HAT metabolic selection, hybridomasupernatants were tested by FACS using human MCF7 breast cancer cells.Only MCF7 binding antibodies were kept.

Antibodies of interest were then cloned by limit dilution. Eight daysafter cloning, supernatants were selected once again by FACS using MCF7cells. Three positive clones were kept for each hybridoma. Isotyping ofthe secreted antibodies is determined using SBA clonotyping system-HRPkit from Southern Biotechnologies (Cat: 5300-05). Finally, one clone isexpanded and frozen.

Further characterizations of 810D12 antibody were then performed usinghybridoma supernatants such as rhIGF-1R or rmIGF-1R or rhIR ELISA. Inall direct ELISAs, proteins of interest were immobilized (1 μg/ml) tothe bottom of each well. After saturation, hybridoma supernatants wereadded to the wells. After a 1-hour incubation period and a washing step,a solution of goat anti-mouse IgG-HRP labelled polyclonal antibody wasused for detection, prior to the addition of the TMB substrate. Thereaction was stopped with a 1M H₂SO₄ solution before reading the OD witha spectrophotometer at a 450 nm wavelength. Data are presented in thefollowing Table 6.

TABLE 6 OD values obtained at 5 μg/ml by ELISA rhIGF1R coating rmIGF1Rcoating rhIR coating 810D12 2.136 0.053 0.048 Positive CTRL 2.338 1.2931.077 Negative CTRL 0.055 0.065 0.048

The dose response curve for the 810D12 antibody on rhIGF-1R coating ispresented in FIG. 1. The values of the EC₅₀ are determined using Prismapplication.

Data showed that the 810D12 antibody only recognizes the rh IGF-1R withan EC₅₀ of 0.51 nM. It does not bind to the murine form of the IGF-1Rnor the human IR.

EXAMPLE 2 Evaluation of the Correlation of the Staging with the Antibodyof the Invention and the Activity of an ADC Targeting IGF-1R in theMCF-7 Xenograft Model

In order to correlate the grading of tumors with the pharmacology, thetumors have been graded (section 2.1) and then in vivo experiments onMCF-7 xenograft model have been made with an ADC comprising an antibodymoiety targeting the IGF-1R known to be internalized and a drug moietyconsisting of an auristatin (section 2.2).

2.1: Immunohistochemistry Detection of the IGF-1R Expression on theMCF-7 Xenograft Model.

Sections of tissue from MCF-7 xenograft were deparaffinized, rehydrated,and placed in Target Retrieval Buffer 1×(Dako S1699) in a boiling bathpre-warm at 98° C. for heat-induced epitope retrieval at 98° C. for 40minutes then 20 additional minutes in the Target Retrieval Buffer. After3 washes in Tris Buffer Saline-0.05% tween 20 (TBS-T) (Dako S3006) theEndogenous peroxidase activity was blocked using Peroxidase BlockingReagent (Dako K4007) for five minutes. Sections were washed with TBS-Tand incubated a blocking reagent (UltraV block-TA-125UB-LabVision) for 5minutes before incubation with either the 810D12 monoclonal antibody (at5 μg/ml) or mouse IgG1/kappa (5 μg/ml, X0931, Dako) as negative controlfor 1 hours at room temperature. Sections were washed with TBS-T andincubated with Envision (Dako) for 30 minutes. Diaminobenzidine was usedfor development of a brown reaction product (Dako K3468). The slideswere immersed in hematoxylin for 2 minutes to counterstain (Dako S3309).

Anti-IGF-1R monoclonal antibody 810D12 of the present inventiondifferentially stains the cell membrane of MCF-7. In this IHC procedure,the brown reaction product correlates to positive staining of the cellmembrane and lack of brown reaction product correlates to negativestaining and no visualization of the cell membrane. Using membranousalgorithm, the scoring for the staining of MCF-7 tumor cells was 3+(FIG. 2A). Using G11 antibody (Roche Ventana) or AF-305 (R&D system)anti-IGF-1R antibodies, section of the same tumor were scored 2+ (FIGS.2B and 2C respectively).

2.2: In Vivo Activity of an Anti-IGF-1R ADC in the MCF-7 XenograftModel.

Anti-IGF-1R ADC has been evaluated in vivo, in the MCF-7 xenograftmodel.

All animal procedures were performed according to the guidelines of the2010/63/UE Directive on the protection of animals used for scientificpurposes. The protocol was approved by the Animal Ethical Committee ofthe Pierre Fabre Institute. Five millions MCF-7 cells were injectedsubcutaneous into 7 weeks old Swiss/Nude mice. Prior to cell injection,oestrogen pellets (Innovative Research of America) were implanted to theleft flank to mice in order to release estrogens necessary to the invivo growth of MCF-7 tumors.

Twenty days after MCF-7 cell implantation, when tumors reached anaverage size of 120-150 mm³, the animals were divided into groups of 6mice according to tumor size and aspect. Anti-IGF-1R ADC was inoculatedby intraperitoneal injections for a 6 injection cycle every four days(Q4d4). The health status of animals was monitored daily. Tumor volumewas measured twice a week with an electronic calliper until study end.Tumor volume is calculated with the following formula:π/6×length×width×height. Toxicity was evaluated following the weight ofanimals three times per week. Statistical analyses were performed ateach measure using a Mann-Whitney test.

Injection of anti-IGF-1R ADC significantly inhibited and even induced acomplete tumor growth regression (FIG. 3) as expected for a tumor graded3+ but not for a tumor graded 2+.

EXAMPLE 3 Evaluation of the Correlation of the Staging with the Antibodyof the Invention and the Activity of an ADC Targeting IGF-1R in theSBC-5 Xenograft Model

In order to correlate the grading of tumors with the pharmacology, thetumors have been graded (section 3.1) and then in vivo experiments onSBC-5 xenograft model have been made with an ADC comprising an antibodymoiety targeting the IGF-1R and a drug moiety consisting of anauristatin (section 3.2).

3.1 Immunohistochemistry Detection of the IGF-1R Expression on the SBC-5Xenograft Model.

Level of IGF-1R was analyzed using the same protocol described insection 2.1 of the example 2 before.

When IGF-1R was detected with the 810D12, low levels were detected (1+).(FIG. 4A). When IGF-1R was detected with G11 antibody (Roche Ventana) orAF-305 (R&Dsystem) anti-IGF-1R antibodies, sections from the same tumorwere scored 3+ (FIGS. 4B and 4C respectively).

3.2: In Vivo Activity of an Anti-IGF-1R ADC in the SBC-5 XenograftModel.

Anti-IGF-1R ADC has been evaluated in vivo, in the SBC-5 xenograftmodel.

All animal procedures were performed according to the guidelines of the2010/63/UE Directive on the protection of animals used for scientificpurposes. The protocol was approved by the Animal Ethical Committee ofthe Pierre Fabre Institute. Five millions SBC-5 cells were injectedsubcutaneous into 7 weeks old Athymic mice. Twelve days after cellimplantation, when tumors reached an average size of 150 mm³, theanimals were divided into groups of 6 mice according to tumor size andaspect. Anti-IGF-1R ADC was inoculated by intraperitoneal injections fora 6 injection cycle every four days (Q4d6). The health status of animalswas monitored daily. Tumor volume was measured twice a week with anelectronic calliper until study end. Tumor volume is calculated with thefollowing formula: π/6×length×width×height. Toxicity was evaluatedfollowing the weight of animals three times per week. Statisticalanalyses were performed at each measure using a Mann-Whitney test.

Tumor progression of SBC-5 tumoral cells was not affected by injectionof anti-IGF-1R ADC. (FIG. 5) as expected for a tumor graded 1+ but notfor a tumor graded 3+.

The invention claimed is:
 1. An IGF-1R antibody, or an antigen-bindingfragment thereof, said antibody comprising: i) a heavy chain comprisingthe following three CDRs, respectively CDR-H1 of sequence SEQ ID No. 1,CDR-H2 of sequence SEQ ID No. 2 and CDR-H3 of sequence SEQ ID No. 3; andii) a light chain comprising the following three CDRs, respectivelyCDR-L1 of sequence SEQ ID No. 4, CDR-L2 of sequence SEQ ID No. 5 andCDR-L3 of sequence SEQ ID No.
 6. 2. The IGF-1R antibody according toclaim 1, said antibody comprising a heavy chain variable domain ofsequence SEQ ID No. 7, or any sequence with at least 90% of homologywith the sequence SEQ ID No. 7; and/or a light chain variable domain ofsequence SEQ ID No. 8, or any sequence with at least 90% of homologywith the sequence SEQ ID No.
 8. 3. An IGF-1R antibody secreted by thehybridoma deposited at the CNCM, Institut Pasteur, Paris, on Sep. 17,2014, under number 1-4893.
 4. A method for detecting in vitro or ex vivothe presence and/or the location of IGF-1R expressing tumoral cells in asubject, said method comprising the steps of: (a) contacting abiological sample from said subject with the IGF-1R antibody, or anantigen-binding fragment thereof, of claim 1; and (b) detecting thebinding of the said IGF-1R antibody, or an antigen-binding fragmentthereof, to said biological sample.
 5. A method for detecting in vitroor ex vivo the percentage of tumoral cells expressing IGF-1R in asubject, said method comprising the steps of: (a) contacting abiological sample from said subject with the IGF-1R antibody, or anantigen-binding fragment thereof, of claim 1; and (b) quantifying thepercentage of cells expressing IGF-1R in said biological sample.
 6. Amethod for determining in vitro or ex vivo the expression level ofIGF-1R in tumoral cells in a subject, said method comprising the stepsof: (a) contacting a biological sample from said subject with the IGF-1Rantibody, or an antigen-binding fragment thereof, of claim 1; and (b)quantifying the level of binding of the said IGF-1R antibody, or anantigen-binding fragment thereof, to IGF-1R in said biological sample.7. A method for determining in vitro or ex vivo the IGF-1R scoring oftumoral cells or of a tumor in a subject, said method comprising thesteps of: (a) contacting a biological sample from said subject with theIGF-1R antibody, or an antigen-binding fragment thereof, of claim 1; (b)quantifying by Fluorescence Activated Cell Sorting (FACS) orimmunohistochemistry (IHC) the level of binding of the said IGF-1Rantibody, or an antigen-binding fragment thereof, to IGF-1R in saidbiological sample; and (c) scoring the tumoral cells or the tumor bycomparing the quantified level obtained in step (b) to an appropriatescale based on the intensity of the staining and the percentage ofpositive cells.
 8. A method for determining whether an oncogenicdisorder is susceptible to treatment with an inhibitor targeting theIGF-1R pathway, said method comprising the steps of: (a) determining invitro or ex vivo the IGF-1R status of tumoral cells or of a tumor of asubject according to the method of claim 6, and (b) determining that, ifthe IGF-1R status of tumoral cells or the tumor is IGF-1R(+), theoncogenic disorder is susceptible to treatment with an antibody drugtargeting the IGF-1R pathway.
 9. The method of claim 8 wherein saidinhibitor is an IGF-1R antibody.
 10. The method of claim 9 wherein saidIGF-1R antibody is alone, combined or conjugated.
 11. A method fordetermining in vitro or ex vivo the efficacy of a therapeutic regimendesigned to alleviate an oncogenic disorder associated with IGF-1R in asubject suffering from said disorder, said method comprising the stepsof: (a) measuring a first expression level of IGF-1R according to themethod of claim 6 in a first biological sample, said first biologicalsample corresponding to first time point of the said treatment; (b)measuring a second expression level of IGF-1R according to the method ofclaim 6 in a second biological sample, said second biological samplecorresponding to a second, later time point of the said treatment; (c)calculating the ratio of said first expression level of step (a) to saidsecond expression level of step (b); and (d) determining that theefficacy of said therapeutic regime is high if the ratio of step (c) isgreater than 1; or determining that the efficacy of said therapeuticregime is low if the ratio of step (c) is inferior or equal to
 1. 12. Amethod for selecting a cancer patient predicted to benefit or not fromthe administration of a therapeutic amount of an inhibitor targeting theIGF-1R pathway, said method comprising the steps of: (a) measuring theexpression level of IGF-1R according to the method of claim 6 in abiological sample of said patient; (b) comparing the expression level of(a) with a reference expression level; and (c) selecting the patient asbeing predicted to benefit from a treatment with an antibody drugtargeting the IGF-1R pathway, if the ratio of the expression level of(a) to said reference expression level is greater than 1; or (d)selecting the patient as being not predicted to benefit from a treatmentwith an antibody drug targeting the IGF-1R pathway, if the ratio of theexpression level of (a) to said reference expression level is inferioror equal to
 1. 13. The method of claim 12 wherein said inhibitor is anIGF-1R antibody.
 14. The method of claim 13 wherein said IGF-1R antibodyis alone, combined or conjugated.
 15. The method of claim 13 whereinsaid reference expression level is the expression level of IGF-1R in abiological sample from a healthy subject.
 16. A kit for the detection ofIGF-1R expressing tumoral cells in a patient, said kit comprising theIGF-1R antibody, or an antigen-binding fragment thereof, of claim
 1. 17.The kit of claim 16, wherein said antibody or antigen-binding thereof islabelled.
 18. The kit of claim 16, further comprising positive andnegative control samples for the scoring of IGF-1R expression level. 19.A murine hybridoma capable of secreting an IGF-1R antibody or anantigen-binding fragment thereof, said murine hybridoma being depositedat the CNCM, Institut Pasteur, Paris, on Sep. 17, 2014, under numberI-4893.
 20. An isolated polynucleotide comprising a sequence coding foran antibody, according to claim
 1. 21. The isolated polynucleotide ofclaim 20, said polynucleotide comprising a sequence selected from thegroup consisting of: SEQ ID NO. 9 and SEQ ID NO.
 10. 22. A vectorcomprising the polynucleotide of claim
 20. 23. A host cell transformedby the vector of claim
 22. 24. A method for producing an IGF-1Rantibody, said method comprising the steps of: a) growing the host cellof claim 23 in a culture medium and under suitable culture conditions;and b) recovering said antibody produced from the culture medium or fromsaid cultured cells.